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Definitions

• IDAAT immune defense activated antithrombin
• Antithrombin III is an important physiological anticoagulant which, without requiring prior activation, inhibits circulating serine proteases.
• the protease cleaves the arginine 393-serine 394 bond, which leads to a change in the conformation of antithrombin and to protease inhibitor complex formation.
• Heparin significantly accelerates the formation of antithrombin-protease complexes by binding in the amino-terminal region of antithrombin III. It is believed that in vivo glycosaminoglycans such as heparan sulfate on the endothelial surface assume the role of heparin.
• Antithrombin III is a member of the serine protease inhibitor (Serpine) family of over 100 members and is a glycoprotein. Its 432 amino acid polypeptide chain has a molecular weight of 58000. The protein contains three intramolecular disulfide bridges and four glycosylation positions. When administered in extremely high, unphysiological doses, antithrombin III reduces the mortality of sepsis in animal experiments (Dickneite and Paques, 1 993). In people with sepsis, however, no significant improvement in mortality or morbidity could be achieved with commercially available antithrombin III preparations.
• Antithrombin III is changed in its form by inflammation-mediated processes.
• the so-called “natural”, “innate” or “constitutive” immune defense is the first defense strategy against "invaders” such as bacteria, viruses, parasites etc. and is widespread throughout the animal kingdom.
• An important part of this first defense is that phagocytotic cells, in particular monocytes and PMNL (neutrophil granulocytes), but also dendridial cells, eosinophils, platelets and mast cells alone, or as associates with other cells migrate to the site of the pathogen's invasion (chemotaxis) and penetrate through epithelia and endothelium (diapedesis).
• the "foreign cells / intruders" are neutralized by phagocytosis.
• the inflammatory cells release proteases such as elastase and cathepsin G and metalloproteases and substances that cause oxidation of lipids, proteins and peptides.
• Halogenation (mainly chlorination) is an important way to kill cells. In the area of inflammation, the pH value is lowered to below pH 4.0 by the release of lactic acid.
• the defense cells also release specific proteins and peptides for defense, such as "bactericidal / permeability-increasing" (BPI) protein from platelets and granulocytes and defensins from granulocytes.
• BPI Bacillicidal / permeability-increasing
• thrombin If there is a wound or other activation of the hemostasis, thrombin, factor Xa and other serine proteases arise.
• complement alternative route, properdinpathway
• Activated mast cells release soluble heparin proteoglycan, which can bind to antithrombin (Linstedt et al., 1 992).
• Antithrombin III is changed indirectly or directly by these processes and receives completely new functions.
• antithrombin III which is directly or indirectly changed by these processes, receives completely new functions.
• antithrombin III can also be converted into this activated form in vitro, in particular by processes such as oxidation, treatment with urea and guanidine hydrochloride, proteolytic cleavage, heating to 60 ° C., lowering the pH to 4.0 or Addition of an ATIII peptide that contains the sequence SEAAAS.
• a cryptic sequence of the antithrombin is exposed and allows the protein to interact with proteins such as thrombospondin, vitronectin, CD36, oxLDL, a> 5 - integrin and others.
• TSP-1 activated antithrombin III
• TSP-1 The local TSP-1 concentration increases more than 1000 times (Flicker and Kehrel, 1 993). Endothelial cells, smooth muscle cells, glial cells and leukocytes also secrete TSP-1.
• TSP-1 is a member of the thrombospondin family, which also includes TSP-2, TSP-3, TSP-4 and the cartilage oligomeric matrix protein (COMP) (Lawler et al., 1 993).
• TSP-1 and TSP-2 are identical in some areas, so that some TSP-1 functions can also be performed by TSP-2. TSP-1 and TSP-2 have the same domain structure and can be expressed as homo- and heteromers (Bornstein et al., 1 991).
• TSP-1 is a trimeric glycoprotein with an apparent mass of 420,000 Da. Its 3 subunits have a molar mass of 1 80000 Da in the Lämmli SDS-PAGE system (Lawler and Hynes 1 986). Electron microscopic images show the trimeric structure, which looks like a bola with globular ends at the amino and carboxy termini of the polypeptide chains (Galvin et al. 1 985). The three chains are linked by disulfide bridges near the globular amino termini. Each TSP-1 subunit contains 69 cysteine residues, so that each chain has at least one free SH group.
• TSP-1 and TSP-2 contain similar functional domains, such as the N-terminal region, a pro-collagen homologous region, type 1 TSP “repeats” (repetitive areas), type 2 TSP “repeats”, type 3 calcium-binding "repeats "and the carboxy-terminal region (Bornstein et al., 1 992).
• the rod-shaped connecting regions of the TSP-1 chains show a calcium-dependent structure. In the presence of Ca 2 + , this structure has a length of 16 to 29.1 nm after EDTA treatment, on the other hand 38.3 nm (Lawler 1 986).
• TSP-1 The conformation of TSP-1 is strongly dependent on the Ca 2+ concentration (Lawler et al. 1 988) and on the binding to interaction partners.
• the binding of TSP-1 to fibronectin or heparin gives it a conformation in the absence of Ca 2+ , which the molecule would adopt in the presence of Ca 2+ (Dardik and Lahav 1 999).
• TSP-1 Immobilized TSP, adsorbed on surfaces, mediates the adhesion of endothelial cells, smooth muscle cells and monocytes. This adhesion depends on the Ca 2+ conformity of the TSP-1. EDTA treatment irreversibly inhibits this process (Lawler et al. 1 988).
• the Ca 2+ form of TSP-1 enables binding to cells RGD-mediated via integrins. Binding to CD36 also causes a change in conformation in the TSP-1 molecule (Leung et al 1 992).
• TSP-1 binds to CD36 through a two-step mechanism. Only in the second step does TSP-1 bind with high affinity to CD36 via the "cell-binding site" in the Properdin-like "Type 1 repeat".
• Binding to CD36 via peptide sequence 1 39-1 55 of CD36 enables a conformational change in TSP-1, which allows high affinity binding to sequence 93-1 10.
• sequence of CD36 In this area lies the sequence of CD36, the binding capacity of which is regulated by phosphorylation / dephosphorylation (Thr 92) (Asch et al., 1 993).
• Thr 92 phosphorylation / dephosphorylation
• TSP-1 The conformation of TSP-1 regulates its functionality.
• TSP-1 deficient mice develop between the 1st and 4th week of life extensive acute and chronic o rganized bacterial pneumonia with massive immigration of neutrophils and macrophages. Diffuse alveolar bleeding has been observed. At a later time of infection, compared to control mice of the same inbred strain that have TSP-1, the epithelium of the airways becomes thickened and crimped (Lawler et al. 1 998).
• TSP-1 negative mice produced significantly less offspring than control animals.
• TSP-1 "knock outs” show a pronounced lordotic curvature of the spine. This shows the importance of TSP-1 for the development / stabilization of the skeleton.
• TSP-1 deficient animals had a highly significant higher number of leukocytes, especially monocytes and eosinophils in the periphery Blood.
• TSP-1 is a multifunctional protein. Immobilized on surfaces, it promotes the formation of plasmin (Silverstein et al. 1 986) and simultaneously protects the plasmin from inactivation by the alpha2 plasmin inhibitor.
• the urokinase plasminogen activator (uPA) and the "signal chain" uPA (scuPA) bind to immobilized TSP-1 and remain proteolytically active.
• the binding to immobilized TSP protects uPA from inhibition by the plasminogen activator inhibitor type 1 (PAI- 1) (Silverstein et al., 1,990).
• scuPA binds to its receptor (scuPAR)
• scuPAR cell-associated TSP-1 and vitronectin (Vn)
• Vn vitronectin
• Immobilized TSP-1 enables proteolytic processes even in a microenvironment in which there is no fibrin.
• immobilized TSP-1 activates the "latent" transformation rowth facto beta 1 (TG F-M) on the macrophage surface (Yehualaeshet et al., 1 999).
• TSP-1 also activates the TGF- /? On the endothelial surface. (Schultz-Cherry and Murphy-Ullrich, 1,993, Schultz-Cherry et al., 1,994). TGF /? inhibits the proliferation of endothelial cells and has an antiangiogenic effect. Inhibition of angiogenesis by TSP-1 has been widely described (Iruela-Arispe et al., 1 999, Jiminez et al., 2000). A complex formation between TSP and FGF-? 1 (basic fibroblast growth factor) is also involved in this function (Murphy-Ullrich, 1 993). Absence of TGF- /?
• TGF- /? deficient animals additionally showed a strong autoimmune reactivity (Letterio et al., 1 996) by influencing MHC class II antigen expression (Geiser et al., 1 993).
• TSP-1 TGF- /? can activate, that TSP-1 over TGF- /? is involved in the described processes of infection control and autoimmune reactivity (Crawford et al., 1 998). Immobilized TSP-1 regulates together with TGF- /? the proliferation of "natural killer" cells (NK cells) (Pierson et al., 1 996). The TSP-1 deficient animals show corresponding immunodeficiencies, albeit to a lesser extent. Since the activated antithrombin described for the first time in this invention , which binds TSP-1, can immobilize TSP on cell surfaces, suggests that IDAAT indirectly influences the activation of TGF-ß. TSP-1 also modulates immunological defense-relevant processes via other mechanisms.
• a large number of microorganisms such as, for example, coagulase-negative staphylococci (Li et al., 2000), enterococci and Porphyromonas gingivalis fimbriae (Nakamura et al., 1 999) adhere to immobilized TSP-1.
• the parasite itself has a membrane protein that contains TSP-1 homologous areas. Via this protein TRAP (thrombospondin-related-anonymus (adhesive) protein) transported into the erythrocyte membrane, the parasite can mediate the attachment of the infected erythrocytes to the vessel wall (Wegelnik et al., 1 999, Kap et al., 1 999).
• TRAP thrombospondin-related-anonymus (adhesive) protein
• pathogens such as Cryptosporidium parvum or Eimeria tenella
• TSP or TSP receptor homologous domains that they use for cell adhesion (Sulaiman et al., 1 999).
• the HIV-1 virus uses in its surface protein GP 1 20 a CD36 (TSP receptor) domain with which the HIV virus can bind to both TSP and CD4 on the host cells (Crombie et al., 1998). Purified TSP-1 can therefore inhibit H1V-1 infections (Crombie et al., 1 998).
• CD36 CD36 receptor
• TSP thrombospondin
• thrombospondin Several complement proteins, C9, C8 alpha and C8 beta, have modules with high homology to one of the "repeat” modules of thrombospondin (Patthy, 1 988).
• Other TSP homologous domains also have antistasin, properdin and F-spondin.
• F-spondin like thrombospondin itself, is an effective substance for improving lesions of the nervous system (US 5750502).
• PMNL apoptotic neutrophil granulocytes
• macrophages TSP can mediate the phagocytosis of apoptotic PMNL.
• TSP can mediate the phagocytosis of apoptotic PMNL. For this process, the simultaneous interaction of TSP with its receptors ⁇ v /?
• phagocytosis of apoptotic PMNL regulates inflammatory reactions and prevents an uncontrolled overshoot.
• TSP-1 mediated phagocytosis of apoptotic PMNL occurs without the release of pro-inflammatory mediators (Stern et al., 1 996).
• TSP-mediated phagocytosis prevents an excessive inflammatory reaction.
• the production of pro-inflammatory cytokines is actively inhibited by macrophages which have taken up apoptotic PMNL (Fadok et al., 1 998).
• Cross-linking of TSP receptor CD47 on monocytes by TSP-1 additionally leads to an inhibition of the release of active interleukin 1 2 (IL-1 2) (Armant et al., 1 999, Demeure et al., 2000).
• Interleukin-1 2 is an important mediator of sepsis (Steinhauser et al., 1 999).
• TSP-1 TSP-1 on apoptotic PMNL and on monocytes is therefore a good way to positively influence medicinal conditions with persistent, chronic inflammation.
• diseases also include all in which an excessive inflammatory reaction is part of the disease itself, such as the various reperfusion damage, rejection reactions in organ transplants and diseases of the rheumatic type.
• TSP immobilization on the cell surface is also a way to combat the unwanted, pro-inflammatory response in diseases that are eosinophil-mediated, such as allergy, asthma, parasitic diseases, certain tumors and connective tissue diseases. This prevents highly toxic substances from being released by the eosinophils and damaging or destroying the tissue.
• TSP binds chemokines like RANTES, thus preventing the chemokine from binding to its receptor (Barnes et al., 1 998). In this way too, TSP modulates inflammatory reactions and immune defense.
• a drug that affects the function of TSP by changing its conformation or promoting immobilization on cell surfaces of immunocompetent cells limits the unwanted immune response in diseases such as, but not limited to, rheumatoid arthritis, goodpasture syndrome, insulin-dependent diabetes , Pemphigus, pemphigoid, primary biliary cirrhosis, ulcerative colitis, lupus erythematosus, graft-versus-host disease, sepsis.
• CLL cells chronic lymphoid leukemia cells
• a substance that causes TSP to bind to leukemia cells would thus be an effective drug in the fight against CLL, a fatal disease for which there is as yet no specific effective drug.
• Thrombospondin not only inhibits via its effect on TGF- /? Neoangiogenesis, but also via immobilization and binding to and activation of CD36.
• Treatment of tumors in mice with TSP-1 leads to inhibition of neoangiogenesis and apoptosis of endothelial cells (Jiminez et al., 2000).
• Inhibiting tumor angiogenesis is a good way to limit the growth of tumors with drugs (Roberts et al.,. 1 996).
• a substance that mediates the binding of TSP to endothelial cells in tumors thus gains antiangiogenic and thus cancer-fighting properties.
• Neoangiogenesis is also a cause of blindness, e.g. due to diabetes mellitus (Kaplan et al., 1 999, Shafiee et al., 2000), age-related macular degeneration or early maturity of infants.
• a substance that mediates the binding of TSP to endothelial cells could also be used with medication to combat this neoangiogenesis.
• TSP concentrations of TSP in the tissue around the injured region increase significantly.
• balloon cathetization e.g. can be detected on the surfaces of the cells TSP as early as 1 hour after injury (Watkins et al., 1 990, Munjal et al., 1 990).
• the TSP on the cell surface increases in the following days and is then also increasingly enriched in the matrix.
• TSP disappears from the cell membrane of the injured tissue.
• TSP wound healing
• US Patent 5, 155, 038 A substance that immobilizes TSP-1 in the wound should improve wound healing.
• TSP in the wound is expressed by the tissue cells but also by platelets, which are released during their activation. About 1% of the total platelet protein and approx.% Of the protein content of the platelets ⁇ -granules is thrombospondin-1 (Kehrel, et al., 1 996). Released thrombospondin promotes collagen-induced platelet aggregation (Kehrel, et al., 1 988). In the C-terminus, TSP contains a sequence, RFYVVMWK, which activates platelets via CD47 (Chung et al., 1 999 and 1 997). Soluble TSP, to which blood, platelet suspension or platelet-rich plasma is added, does not trigger aggregation alone.
• platelets in suspension TSP only can bind Ca 2+ form in his, platelets adhere to the immobilized matrix thrombospondin both the "high” and “low” Ca 2 + form.
• the object of the present invention was therefore to provide a medicament which can perform the functions mentioned above and thus fulfill the expected effects.
• activated IDAAT triggers or mediates a large number of reactions in the body which can be used for the treatment of diseases via its properties and functions which have now been ascertained within the scope of the present invention. These functions and properties will be explained in the following, as well as the diseases or disease states to be treated with them. In many cases, prophylaxis can also take place with the help of IDAAT.
• the medicament can contain both the complete IDAAT, which can be produced, for example, according to the method described in the examples.
• Theoretically can also be isolated from the body after defense reaction IDAAT.
• IDAAT peptides which mediate the interaction with proteins such as thrombospondin, vitronectin, CD36, oxLDL,
• suitable peptides can easily be found by preparatory experiments, for example by testing their interaction with one of the above-mentioned proteins.
• Analogs of IDAAT are also suitable in the context of the present invention if they also mediate the interaction with the proteins mentioned. Finally, it is also possible to use IDAAT mimetics which, due to their structure and / or functional groups, can show the same effects and interactions as IDAAT.
• recombinant IDAAT for which purpose recombinantly produced antithrombin III is treated in a suitable manner in order to obtain activated antithrombin III (see examples and this description).
• Peptides or analogs of IDAAT are also preferably synthesized in recombinant form and then activated.
• a medicament according to the invention can of course contain further pharmaceutically acceptable auxiliary or / and carrier substances, the medicament being formulated for local, intradermal, superficial, intraperitoneal, intravenous or intramuscular or oral administration or its administration being made possible via vesicles.
• the medicinal product according to the invention therefore preferably contains auxiliary substances and carrier substances which are made possible by the preferred type of application.
• the medicament according to the invention can contain further substances, such as For example, antibiotics, immunosuppressive drugs, etc. Depending on the illness to be treated, it can be advantageous to treat with known medicinal products. A corresponding combination of this medicament with IDAAT or its analogs is therefore possibly a preferred embodiment of the present invention.
• the medicament according to the invention can be used for a large number of indications.
• the following are examples of new functions of IDAAT that do not have antithrombin preparations and, in particular, commercial antithrombin preparations:
• IDAAT mediates specific and dose-dependent binding of TSP-1 to monocytes, monocytic cell lines and monocytic cells such as macrophages.
• IDAAT mediates the TSP connection to apoptotic PMNL PMNL made apoptotic by aging (24 h incubation in cell culture medium in the incubator according to Savill 1 992) bind TSP. This process is increased in a dose-dependent, specific manner by adding IDAAT (see Figure 2). Simultaneous addition of purified TSP + IDAAT further increases the effect.
• IDAAT promotes the transmigration of monocytes through the endothelium depending on the dose
• TSP + IDAAT results in low
• IDAAT induces Ca 2+ flux in monocytes.
• the Ca 2+ measurement was carried out according to Sorrani et al., 1 993. Eluted monocytes (5x1 0 6 / ml) were washed at room temperature with Hepes-Tyrode buffer pH 7.4 and then labeled for 1 5 min with 1 ⁇ M Fura2 / AM at 37 ° C, washed twice in Hepes-Tyrode buffer without Ca 2+ and then in Hepes-Tyrode with 1 mM Ca 2+ .
• Ca 2+ signals induced by IDAAT, ' TSP peptide RFYVVMWK, and substances which act as positive or negative controls were determined fluorometrically in the Hitachi F-2000.
• IDAAT (1 00 / g / ml) activates the monocytes and produces a clear Ca 2 + signal (see Figure 5).
• IDAAT mediates the connection of TSP-1 to T cells and to dendridial cells
• IDAAT mediates the connection of T-cell secreted TSP and of exogenously added TSP to human T-cells (here as an example Jurkat cells) (see Figure 6).
• IDAAT increases the activating effect of fMLF on the oxidative burst of PMNL in a dose-dependent manner
• the induction of the oxidative burst was carried out essentially according to the manufacturer's instructions using the phagotest / burst test from Orpegen (Heidelberg) on the flow cytometer, but first the PMNL was incubated with the substrate DHR1 23 and then the PMNL was activated.
• IDAAT increases the activating effect of fLMF on the oxidative burst.
• IDAAT not only increases the activating effect of other agonists on the oxidative burst of PMNL, but also triggers it as an independent agonist (see Figure 7).
• IDAAT is a valuable tool for increasing the defense against infections.
• IDAAT specifically and dose-dependent inhibits the release of active interleukin 12 (IL-12) by activated monocytes
• Active 1L-1 2 plays a key negative role in inflammatory reactions and sepsis.
• Monocytes produce and release IL-1 2.
• This reaction could be inhibited by IDAAT in a dose-dependent manner.
• the concentration of active 1L-1 2 in the culture medium of the monocytes was determined by means of ELISA.
• the IL-1 2 release was completely inhibited by incubating the monocytes with IDAAT (see Figure 8).
• mice were additionally injected intraperitoneally in buffer at 0 and after 0 + 3 hours each with 50 ⁇ g IDAAT in buffer.
• IDAAT inhibits HIV-1 infection of peripheral blood monocytic cells (PBMC)
• PHA-activated PBMC were combined with negative human serum 1: 1 00 (negative control), with neutralizing V3ioop-specific antibody (positive control), with IDAAT (1 50 ⁇ g / ml) and a CCR5 tropic HIV-1 primary isolate (903) incubated from a patient and after five days the virus production was examined using p24-ELISA.
• freshly PHA-activated PBMC were taken up in RPMI 1 640 medium + 20% FCS + 100 U / ml IL-2 in a cell concentration of 2x10 6 cells / ml and with 200,000 cells / well / 1 00 ⁇ ⁇ each on a 96-well Flat bottom plate distributed.
• Positive control neutralizing human anti V3loop antibody 1: 100,
• Negative control negative human serum 1: 100 and
• Verum IDAAT (150 ⁇ g / ml) were added to the cells in RPM1 medium and incubated for 30 minutes at 37 ° C./5% CO 2 .
• the HIV-1 virus was then added to the batches: in each case 10 ⁇ l / well of HIV-1 primary isolate 903 supernatant (CCR5-trop) with 20,000 TCID 50 (50% tissue culture infective dose) / ml s 1000 TCID 50 / ml in the well.
• P24-ELISA The ⁇ -p24 antibodies used (1 1 -G7 [Low, Berlin] and D7320 [Biochrom]) recognize the p24 protein of the primary isolate variant 903 used. Maxi Sorb ELISA plates (Nunc) were coated with these antibodies overnight , The virus supernatant from the inhibition experiment was inactivated with 1% Triton X-100. The inactivated virus supernatant and the alkaline phosphatase-conjugated detection antibody (BC1 071-AP [Aalto]) were, after washing the coated wells with PBS, transferred together into the wells and incubated there for 5 hours at 37 ° C.
• BC1 071-AP [Aalto] alkaline phosphatase-conjugated detection antibody
• the wells were washed again with PBS, dissolved substrate for the alkaline phosphatase p-nitrophenyl phosphate [Sigma] was added to the wells and the color development after 20 minutes at 405 nm was measured in an ELISA photometer.
• the HIV-1 infection of the PBMCs was effectively inhibited by adding IDAAT.
• IDAAT mediates S. aureus binding to cells capable of phagocytosis and defense against bacteria (platelets, monocytes, PMNL)
• Platelets were labeled with a platelet-specific phycoerythrin-conjugated anti GPIX antibody (clone Beb 1).
• Bacteria (various S.aureus strains) were adjusted to a number of 250,000 germs / ⁇ l with Tris-buffered saline solutions (TBS) and labeled with a RNA dye Syto 13 [MoBiTec, Göttingen] in a concentration of 2 ⁇ M. Labeled bacteria and labeled platelets were co-incubated in a ratio of 1 0: 1 for 10 minutes.
• IDAAT mediates the binding of thrombospondin to platelets.
• Purified TSP-1 was labeled with FITC and added at a concentration of 50 ⁇ g / ml gel-filtered platelets (50,000 / ⁇ l) in Hepes-Tyrode buffer with BSA.
• IDAAT was added in increasing concentrations and incubated together with the platelets at room temperature for 60 minutes. Bound TSP-FITC on the platelet surface was quantified in the flow cytometer.
• IDAAT mediated the binding of thrombospondin to platelets (see Figure 1 3).
• IDAAT promotes fibrinogen binding to platelet gel-filtered platelets 50,000 / ⁇ l in Hepes-Tyrode BSA buffer or plasma rich in platelets anticoagulated with PPACK (50,000 / ⁇ l) was added to FITC-conjugated fibrinogen (1 50 ⁇ g / ml).
• the platelet suspensions were treated with meth. Type I collagen, as described in Kehrel et al., 1 998, activated. Some of the samples were mixed with IDAAT in increasing concentrations. After incubation for 30 minutes at room temperature, the platelets were fixed, washed and the fibrinogen binding was determined quantitatively in the flow cytometer.
• IDAAT increases the fibrinogen binding to platelets induced by collagen activation (see Figure 1 4a).
• I DAAT promotes the adhesion of platelets to adhesion proteins such as thrombospondin, fibrinogen, fibronectin, vitronectin and collagen.
• Platelet adhesion was performed according to Santoro et al., 1 994. Microtiter plates (96 well) were "coated” with adhesion proteins in a concentration of 25 ⁇ g / ml overnight at 4 ° C. and the plates were blocked with BSA. 100 ⁇ l gel-filtered plates or platelet-rich plasma anticoagulated with hirudin (300,000 Plt / ⁇ l) were used Incubated in a moist chamber in the wells at room temperature for 1 hour. Non-adherent platelets were thoroughly washed out. The number of adherent platelets was determined by lysing the platelets with Triton X-100 and detection of the lysosomal enzyme hexosaminidase.
• the increase in platelet adhesion by IDAAT is an integrin (a y ß z , -7llby-? 3) and CD36-mediated reaction (see Figures 1 6 and 1 7).
• the platelet adhesion mediated by IDAAT is not thrombin-mediated and therefore also takes place in the blood anticoagulated with hirudin (see Figure 1 8).
• Heparan sulfate (0-1 0 ⁇ g / ml) and sulfatide (0-20 ⁇ g / ml) do not inhibit the adhesion mediated by IDAAT.
• IDAAT mediated platelet adhesion is dependent on divalent ions. 5 mM EDTA completely inhibit this adhesion to thrombospondin and collagen (see Figure 1 9). 20 uM Mg 2+, 1 mM Ca 2+ or other divalent ions increase the IDAAT mediated platelet adhesion to collagen.
• Soluble TSP-1 inhibits IDAAT-mediated platelet adhesion to collagen or immobilized TSP-1 (see Figure 20). Adding monocytes to platelets increases platelet adhesion to thrombospondin and collagen, while adding red blood cells inhibits platelet adhesion to TSP and collagen (see Figure 21).
• the platelet adhesion to thrombospondin mediated by IDAAT can be completely inhibited by the PI-3 kinase inhibitors Wortmannin and LY294002 (see Figure 22).
• IDAAT mediates the TSP-mediated aggregation of platelets.
• Gel-filtered platelets 200,000 / ⁇ l
• Hepes-Tyrode buffer pH7.4 with the addition of fibrinogen (100 ⁇ g / ml) were examined in the Born aggregator. While purified thrombospondin (25 ⁇ g / ml) alone did not induce aggregation, the addition of IDAAT led to the aggregation in a dose-dependent manner, which was significantly increased by simultaneous administration of TSP and IDAAT (see Figure 23).
• IDAAT mediates the formation of platelet microparticles.
• Gel-filtered platelets were activated with the TSP-1 peptide RFYVVMWK (40 ⁇ M).
• the microparticle formation was marked with a platelet-specific anti GPIX phycoerytrin-conjugated antibody according to Dörmann et al., 1 999, measured in the flow cytometer. Addition of IDAAT, depending on the dose, led to microparticle formation of the activated platelets (see Figure 24).
• the platelets were saturated with a FITC-conjugated monoclonal antibody against the platelet-specific antigen GPIX (clone Beb 1) and the monocytes with a PE-conjugated monoclonal antibody against CD1 4 (clone: M ⁇ P9) Concentrations marked after cell activation and cell fixation. The association was quantified by detection of CD14 and GPIX positive particles. The percentage of leukocytes associated with platelets was related to the total leukocyte population. Platelets (25,000 / ⁇ l) and monocytes (3000 / ⁇ l) were incubated together for 30 minutes. The platelets were previously incubated for 30 minutes with IDAAT and then washed. IDAAT increased the association rate from 11.7% to 7.3% (5 ⁇ g / ml IDAAT) or 20.5% (10 mg / ml IDAAT).
• GPIX platelet-specific antigen
• HMEC-1 Human microvascular endothelial cells (HMEC-1) (3000 / ⁇ l) in RPMI 1 640 medium were incubated for 30 minutes at room temperature with IDAAT or TSP-1 (25 ⁇ g / ml) plus IDAAT. The endothelial cells were fixed, washed and TSP was detected with a monoclonal PE-conjugated anti TSP antibody (clone P10) in the flow cytometer. The median of fluorescence, which reflects the binding of the anti TSP antibody, rose from 79 (without IDAAT) to 1 38 (5 ⁇ g / ml IDAAT).
• Latex beads (3.2 ⁇ m) were coated with active vitronectin (25 ⁇ g / ml) at 4 ° C. overnight and then washed. Vitronectin-containing beads were removed with gel-filtered platelets (25,000 / ⁇ l) and
• Platelets were labeled with anti GPIX (clone Beb 1) and associates from vitronectin-coated beads with platelets in the
• IDAAT, IDAAT-TSP-1 aggregates, commercially available ATIII and commercially available ATIII with TSP-1 addition were represented by electron microscopy using the "rotary shadowing" method according to Jander et al (1 984).
• IDAAT consisted of polymeric ATIII molecules, while commercially available ATIII preparations had a monomeric structure (see Figures 27 a and b). Addition of TSP-1 to IDAAT led to the formation of large IDAAT-TSP-1 complexes, whereas this was not observed with commercially available ATIII (see Figures 27 c and d).
• IDAAT has new protein binding properties
• IDAAT binds directly to proteins to which unactivated antithrombin cannot bind, such as CD4 (e.g. T cells) ( Figure 28), GP1 20 of the HIV virus (Figure 29), thrombospondin (Figure 30), active vitronectin (Figure 31 ), CD36, ⁇ v ß3-integrin.
• CD4 e.g. T cells
• Figure 28 GP1 20 of the HIV virus
• Figure 30 thrombospondin
• active vitronectin Figure 31
• CD36 ⁇ v ß3-integrin.
• the binding of IDAAT to these proteins was determined by means of ELISA on purified or recombinant proteins carried out.
• the purification of TSP-1, active vitronectin, ⁇ llb ß3-integrin and CD36 was carried out as in Kehrel et al. 1,993, Yatohgo et al. 1 988 and Kronenberg, Grahl and Kehrel 1 998.
• IDAAT can be produced, for example, as follows:
• antithrombin III is oxidized with NaOCI and passed over a Sephadex column (Example 1). Before the oxidation, antithrombin III can preferably be incubated with neutrophil granulocyte elastase (see Example 2). Before the oxidation, antithrombin III can also be cleaved with matrix metalloproteinase (see example 3). Antithrombin III can also be activated by reaction with Defensin 2 (see Example 4).
• This process for the production of IDAAT is a further object of the present invention.
• Another object of the present invention is the use of a medicament according to the invention, it being possible to treat the diseases or disease states mentioned below in humans or animals.
• the medicament according to the invention can be used both for prophylaxis and as a remedy.
• Indications are acute infections, in particular infections with pathogens that bind directly or indirectly to IDAAT or an interaction partner, very particularly: the group of HI viruses, parasites such as Plasmodium falciparum and Pneumocystis carinii, and bacteria such as Staphylococcus aureus; Improvement of the immune defense and as a means of prophylaxis of sepsis in patients with a high risk of infection, such as after surgery with a high risk of infection, polytrauma, burns, intoxications, under chemotherapy, in immunosuppressed patients and patients with predisposed immune deficiency; for medicinally influencing acute, chronic or allergic Inflammation reactions, in particular for modulation of inflammatory reactions in which apoptotic PMNL and eos
• Myocardial infarction, constrictions b) organ transplants (prevention of graft rejection) and c) allergic reactions (including neurodermatitis, bronchial asthma).
• the medicament according to the invention can also be used as an additive, e.g. for the mouth, vagina, anus and eyes, as an additive to prevent transmission of infections during sexual contact (such as condoms, diaphragms etc.) and in solutions, plasters and wound dressings for wound care.
• FIG. 1 IDAAT mediates TSP-1 binding to monocytes.
• TSP-1 positive monocytes were labeled with the monoclonal anti TSP antibody clone P10, which was conjugated with phycoerythrin, and the fluorescence of the monocytes was measured in the flow cytometer a) 3000 measured monocytes: monocytes with Hepes-Tyrode buffer pH 7.4 incubated for 30 min; washed and labeled with P1 0-PE b) 3000 measured monocytes: monocytes with TSP-1 (10 ⁇ g / ml) in Hepes-Tyrode buffer pH7.4 30 min, RT incubated; washed and labeled with P1 0-PE c) 3000 measured monocytes: monocytes with TSP-1 (10 ⁇ g / ml) and IDAAT (10 ⁇ g / ml) in Hepes-Tyrode buffer pH7.4 30 min, RT incubated; washed
• FIG. 2 IDAAT mediates the TSP-1 binding to apoptotic polymorphonuclear granulocytes (PMNL)
• PMNL apoptotic polymorphonuclear granulocytes
• a) PMNL were made apoptotic by incubation in cell culture medium for 24 hours in the incubator according to Savill et al., 1,992.
• Apoptotic PMNL were incubated with Hepes-Tyrode buffer pH7.4 for 30 min, washed, labeled with anti TSP AK P10-PE and the fluorescence of 3000 cells was measured by flow cytometry
• PMNL were incubated in cell culture medium for 24 hours in an incubator according to Savill et al ., 1 992 made apoptotic.
• Apoptotic PMNL were treated with TSP-1 (5 ⁇ g / ml) and IDAAT (10 ⁇ g / ml) incubated in Hepes-Tyrode buffer pH7.4 for 30 min, washed, labeled with TSP AK P1 0-PE and the fluorescence of 3000 cells measured by flow cytometry, c) procedure analogous to a and b: IDAAT used in increasing concentrations.
• IDAAT mediates the binding of endogenously present and exogenously added (10 ⁇ g / ml) thrombospondin.
• FIG. 3 IDAAT cross-linked apoptotic PMNL with monocytes by TSP ElutriABLE PMNL and elutri Of monocytes were incubated with TSP and IDAAT in different concentrations for 30 min at RT and the cells were fixed.
• the PMNL were labeled with a monoclonal FITC-conjugated antibody against CD1 6b and the monocytes with anti CD14-PE. PE and FITC positive associations were measured with the flow cytometer.
• FIG. 4 In "Transwell” cell culture chambers (Costar, Bodenheim), a “Transwell” insert covered with a microporous polycarbonate membrane was placed per one of the 24 wells.
• the polycarbonate membrane with a pore size of 5 ⁇ m was coated with fibronectin and then human microvascular endothelial cells (HMEC-1) were cultivated to confluence.
• HMEC-1 human microvascular endothelial cells
• FIG. 5 IDAAT activates monocytes and produces a Ca 2+ signal. The methodology used was described in detail in the description of the example.
• IDAAT mediates the connection of TSP-1 to T cells.
• Cultivated human T cells Jurkat cells
• IDAAT or IDAAT plus TSP-1 in the specified concentrations.
• TSP-1 bound to the T cells was labeled by the monoclonal PE-conjugated anti TSP antibody (clone P10) and measured in a flow cytometer.
• IDAAT mediates the binding of endogenously present and exogenously added TSP to T cells.
• TSP-1 additive 25 ⁇ g / ml
• anti TSP-AK-PE label b) TSP-1 additive (25 ⁇ g / ml); without IDAAT addition; anti TSP-AK-PE label c) TSP-1 additive (25 ⁇ g / ml); IDAAT additive (1 ⁇ g / ml); anti TSP-AK-PE label d) TSP-1 additive (25 ⁇ g / ml); IDAAT additive (5 ⁇ g / ml); anti TSP-AK-PE label e) Plus / minus TSP-1 (25 ⁇ g / ml); IDAAT in increasing concentrations; anti TSP-AK-PE marking
• IDAAT increases the activating effect of fLMF on the oxidative burst of PMNL a) fLMF triggers the oxidative burst of PMNL depending on the dose b) IDAAT increases the oxidative burst triggered by fLMF and is able to induce the oxidative burst in PMNL independently of other agonists
• FIG. 8 IDAAT inhibits the release of active interleukin 1 2 by monocytes activated with interferon y + S. aureus. The methodology used was described in detail in the description of the example.
• Figure 9 The IL-10 secretion of monocytes activated with S. aureus and interferon y is increased by IDAAT.
• IDAAT thus promotes the secretion of an interior vaccine, which protects against LPS-induced lethality in animal experiments.
• the methodology applied was carried out analogously to Figure 8.
• IL-10 was determined by ELISA.
• FIG. 10 The TNF ⁇ r secretion of S. aureus and interferon y-activated monocytes is inhibited by the TSP-1 peptide RFYVVMWK (1 0a). IDAAT increases the inhibitory effect of the TSP-1 peptide (25 ⁇ M) (10b). TNF a was determined by ELISA. The methodology applied was carried out analogously to Figure 8.
• Figure 1 1 IDAAT inhibits the Arthus reaction in the ear of Balb-C mice a) Twice at 0 and 0 + 3 hours, 50 ⁇ g IDAAT i.p. treated mouse. Arthus reaction in the left ear b) Twice control buffer i.p. treated mouse. Arthus reaction in the left ear c) BSA-FITC stored in the ears as a measure of the Arthus reaction in mice treated with IDAAT or control buffer. Arthus reaction in the left ear.
• Figure 12 a) Dot-plot representation of the platelet-bacteria association
• FIG. 13 Purified Ca2 + -containing TSP-1 from human platelets was conjugated with FITC (TSP-1 -FITC) and added to gel-filtered platelets. This mixture was incubated for 1 hour at RT with IDAAT (2 ⁇ g / ml and 5 ⁇ g / ml) and measured in a flow cytometer (5000 platelets)
• Figure 14 a) Gel-filtered human platelets (50,000 / ⁇ l) were treated with 1 50 ⁇ / ml FITC-conjugated fibrinogen and incubated with collagen in increasing concentrations in the absence or presence of IDAAT (5 ⁇ g / ml). After 30 minutes of incubation, the platelets were measured in the flow cytometer. b) Gel-filtered human platelets (50,000 / ⁇ l) were mixed with 1 50 ⁇ / ml FITC-conjugated fibrinogen and IDAAT without TSP or with TSP-1 (10 ⁇ g / ml) added in increasing concentrations. After 1 hour of incubation, the platelets were measured in the flow cytometer.
• Figure 15 a) IDAAT increases the adhesion of platelets to adhesion proteins: fibronectin, vitronectin, fibrinogen, thrombospondin-1 and collagen b) Comparison of IDAAT and commercially available ATIII preparations on the effect of platelet adhesion. The methodology used was described in detail in the description of the example.
• Figure 16 a) IDAAT-mediated platelet adhesion to immobilized thrombospondin-1 is achieved by soluble integrins a ] ib ß 3
• FIG 17 The IDAAT-mediated adhesion of platelets to immobilized TSP-1 is completely inhibited by the CD36-specific antibody clone 37, while the Fc receptor is blocked by IV.3. Blocking the Fc receptor alone has no effect. The methodology used was described in detail in the description of the example.
• Figure 18 IDAAT mediated platelet adhesion was performed with hirudin (20 U / ml) anticoagulated platelet-rich plasma. The methodology used was described in detail in the description of the example.
• FIG. 19 IDAAT-mediated platelet adhesion to TSP-1 is dependent on divalent ions.
• FIG. 20 Addition of soluble TSP-1 inhibits platelet adhesion to collagen in a dose-dependent manner, while IDAAT, which immobilizes TSP-1, increases platelet adhesion to collagen in a dose-dependent manner.
• IDAAT which immobilizes TSP-1
• Figure 21 Addition of monocytes (1 00 / ⁇ l) to platelets (300,000 / ⁇ l) increases the adhesion of platelets to TSP-1, while addition of erythrocytes (20,000 / ⁇ l) has an inhibitory effect.
• the methodology used was described in detail in the description of the example.
• FIG. 22 IDAAT-induced platelet adhesion was inhibited by inhibitors of PI-3 kinase Wortmannin (20 nM) and LY294002 (50 ⁇ M). Wortmannin and LY294002 were pre-incubated for 1 0 min before adding IDAAT.
• Platelet aggregation was carried out according to Born 1 962.
• Addition of IDAAT resulted in a weak aggregation reaction.
• Simultaneous administration of IDAAT and soluble TSP-1 led to strong aggregate formation.
• FIG. 24 IDAAT mediates platelet microparticle formation.
• Gel-filtered platelets (50,000 / ⁇ l) were activated with the TSP-1 peptide RFYVVMWK (40 ⁇ M) and IDAAT was added in increasing concentrations. After 30 minutes of incubation, platelets and microparticles formed from platelets were marked with anti GPIX-PE and the number of microparticles formed was measured in relation to 5000 counted platelets in the flow cytometer.
• FIG. 25 IDAAT mediates TSP-1 binding to endothelial cells. The methodology was described in detail in the description of the example. IDAAT enhances TSP-1 binding to endothelial cells.
• FIG. 26 IDAAT increases the binding of platelets to vitronectin coated latex beads. The methodology was detailed in the description of the example.
• Figure 27 a) IDAAT consists of polymeric ATIII, represented by the "rotary shadowing" electron microscopy method b) Commercial ATIII consists of monomeric globular molecules; electron microscope image after rotary evaporation c) IDAAT (1 mg / ml) and TSP-1 (200 ⁇ g / ml) were incubated for 1 hour at RT.IDAAT and TSP-1 together form large associations; electron microscopic images after rotary evaporation d) commercial ATIII (1 mg / ml) and TSP-1 (200 ⁇ g / ml) were incubated for 1 hour at RT, commercially available AT III and TSP-1 did not react with each other; electron micrograph after rotary evaporation
• Figure 28 IDAAT binds directly to CD4.
• Recombinant CD4 (1 ⁇ g / 100 ⁇ l / well) was bound to the bottom of an ELISA plate (Nunc-Maxisorb).
• the plate was pH 7.4 with PBS, 0.5%.
• Tween 20 washed thoroughly and free spaces on the plastic surface blocked with 3% BSA for 1 hour at room temperature.
• the plate was washed again and then IDAAT or commercially available ATIII in increasing concentrations of 0-5 ⁇ g / ml was added for 1 hour at RT.
• the ATIII solutions were removed, the plate was washed thoroughly and incubated with a polyclonal monospecific antibody against ATIII from the rabbit DAKO, Hamburg) in a dilution of 1: 1500 in PBS, 1% NGS (normal goat serum). The plate was washed again and then incubated with a goat affinity-purified antibody against rabbit IgG conjugated with peroxidase (BIORAD, Kunststoff) at a dilution of 1: 3000.
• the plate was washed again several times and with substrate solution (100 ⁇ l / well) (20 mg ortho-phenyldiamine, 5% H 2 O 2 in a buffer of 1 2, 1 5 ml 0, 1 M citric acid and 1 2.85 ml 0.2 M Na 2 HPO 4 plus 25 ml distilled water) added.
• the absorbance at 405 nm measured in the ELISA photometer reflects the amount of bound antithrombin.
• the reaction was stopped with 50 ⁇ l / well 4 NH 2 SO 4 and the absorbance measured at 490 nm. The reaction clearly shows that IDAAT not only mediates TSP-1, but can also bind directly to CD4.
• Recombinant HIV-GP1 20 (1 ⁇ g / 100 ⁇ l / well) was bound to the bottom of an ELISA plate (Nunc-Maxisorb).
• the plate was pH 7.4 with PBS,
• the plate was washed again several times and with substrate solution (100 ⁇ l / well) (20 mg ortho-phenyldiamine, 5% H 2 O 2 in a buffer of 1 2, 1 5 ml 0.1 M citric acid and 1 2.85 ml 0.2 M Na 2 HP0 4 plus 25 ml distilled water) added.
• substrate solution 100 ⁇ l / well
• the absorbance at 405 nm measured in the ELISA photometer reflects the amount of bound antithrombin.
• the reaction was stopped with 50 ⁇ l / well 4 NH 2 SO 4 and the absorbance measured at 490 nm. The reaction clearly shows that IDAAT not only mediates TSP-1, but can also bind directly to HIV-GP1 20.
• thrombospandin-1 (1 ⁇ g / 100 ⁇ l / well) was bound to the bottom of an ELISA plate (Nunc-Maxisorb). The plate was washed thoroughly with PBS pH 7.4, 0.05% Tween 20 and free spaces on the plastic surface were blocked with 3% BSA for 1 hour at room temperature. The plate was washed again and then IDAAT or commercially available ATIII in increasing concentrations of 0-5 ⁇ g / ml was added for 1 hour at RT. The ATlll solutions were removed, the plate was washed thoroughly and incubated with a polyclonal monospecific antibody against ATIII from the rabbit (DAKO, Hamburg) in a dilution of 1: 1500 in PBS, 1% NGS (normal goat serum).
• the plate was washed again and then with an affinity-purified antibody from goat against rabbit IgG, which with Peroxidase conjugated (BIORAD, Munich), incubated in a dilution of 1: 3000.
• the plate was washed again several times and with substrate solution (100 ⁇ l / well) (20 mg ortho-phenyldiamine, 5% H 2 O 2 in a buffer of 1 2, 1 5 ml 0.1 M citric acid and 1 2.85 ml 0.2 M Na 2 HP0 4 plus 25 ml distilled water) added.
• the absorbance at 405 nm measured in the ELISA photometer reflects the amount of bound antithrombin.
• the reaction was stopped with 50 ⁇ l / well 4 NH 2 S0 4 and the absorbance measured at 490 nm. The reaction clearly shows that IDAAT can bind directly to TSP-1.
• FIG. 31 IDAAT binds directly to Vitronectin (active form) Vitronectin (1 ⁇ g / 100 ⁇ l / well) was bound to the bottom of an ELISA plate (Nunc-Maxisorb). The plate was washed thoroughly with PBS pH 7.4, 0.05% Tween 20 and free spaces on the plastic surface were blocked with 3% BSA for 1 hour at room temperature. The plate was washed again and then IDAAT or commercially available ATIII in increasing concentrations of 0-5 ⁇ g / ml was added for 1 hour at RT.
• the ATIII solutions were removed, the plate was washed thoroughly and incubated with a polyclonal monospecific antibody against ATIII from the rabbit (DAKO, Hamburg) in a dilution of 1: 1500 in PBS, 1% NGS (normal goat serum). The plate was washed again and then incubated with a goat affinity-purified antibody against rabbit IgG conjugated with peroxidase (BIORAD, Kunststoff) at a dilution of 1: 3000.
• the plate was washed again several times and with substrate solution (100 ⁇ l / well) (20 mg ortho-phenyldiamine, 5% H 2 O 2 in a buffer of 1 2, 1 5 ml 0, 1 M citric acid and 1 2.85 ml 0.2 M Na 2 HP0 4 plus 25 ml aqua dest.) Added.
• substrate solution 100 ⁇ l / well
• the absorbance at 405 nm measured in the ELISA photometer reflects the amount of bound antithrombin.
• the reaction was stopped with 50 ⁇ l / well 4 NH 2 SO 4 and the absorbance measured at 490 nm.
• the reaction clearly shows that IDAAT can bind directly to active vitronectin.
• non-functional, non-activated antithrombin III was either obtained from Calbiochem, Sigma, Enzyme Research Laboratories, Pharmacia & Upjohn, Aventis, Baxter or Grifols or was purified from human plasma.
• the antithrombin preparations were buffered against phosphate-buffered saline (PBS) pH 7.4. 348 ⁇ g of pure antithrombin was brought to a volume of 1 ml with PBS pH 7.4 and 0.1 mM EDTA and the solution was cooled on ice.
• Cold NaOCI (832 ⁇ g) was added in a volume of 10 ⁇ l and the mixture was incubated on ice for 10 minutes. The reaction was terminated by immediate gel filtration at 4 ° C over Sephadex G25 (PD 10 columns).
• non-activated antithrombin III was buffered against PBS pH 8.0. 500 ⁇ g of pure antithrombin III was mixed with 50 ⁇ g neutrophil granulocyte elastase (HNE) (human, dissolved in 50 ⁇ l buffer) and the mixture (500 ⁇ l volume) was incubated at 37 ° C. for 16 hours. The reaction was stopped with 1 mM (final concentration) phenylmethylsulfonyl fluoride (PMSF) and the antithrombin III was buffered using the Centricon method against PBS / 0.1 mM EDTA pH 7.4. This was followed by oxidation with NaOCI as described in Example 1.
• HNE neutrophil granulocyte elastase
• PMSF phenylmethylsulfonyl fluoride
• non-activated antithrombin III was buffered against PBS pH 8.0.
• 500 ⁇ g of pure antithrombin III was prepared with 1 2.4 ⁇ g of matrix metalloproteinase-2 (MMP-2) (dissolved in 0.9% NaCl) in 25 mM Tris / HCl / 30 mM NaCl / 10 mM Ca 2+ buffer.
• MMP-2 matrix metalloproteinase-2
• it was pretreated for 2 hours at RT with 1 mM APMA (4-aminophenyl mercuric acetate).
• the batch 500 ul volume was incubated for 1 6 hours at 37 ° C.
• the antithrombin DI was buffered using the Centricon method against PBS pH 7.4.
• Example 4 Commercial, non-activated antithrombin III was buffered against PBS pH 8.0. 200 ⁇ g of pure antithrombin III was incubated with Defensin 2 (HNP-2, dissolved in 0.9% NaCl, final concentration 10 ⁇ M) in PBS pH 8.0 for 1 hour at RT.
• HNP-2 Defensin 2
• Thrombospondin-1 is a major activator of TGF-betal in vivo.
• Fadok VA Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM: Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine / paracrine mechanis s involving TGF-beta, PGE2, and PAF.
• Galvin NJ Dixit VM, O'Rourke KM, Santoro SA, Grant GA, Frazier WA: Mapping of epitopes for monocional antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing. J. Cell Biol. 101: 1434-1 441, 1 985
• TGF Transforming growth factor beta 1
• Beta 1 controls expression o major histocompatibility genes in the postnatal mouse: aberrant histocompatibility antigen expression in the pathogenesis of the TGF beta 1 null mouse phenotype. Proc. Natl. Acad. Sci. USA 90: 9944-9948, 1 993
• Kehrel B Kronenberg A, Schwippert B, Niesing-Bresch D, Niehues U, Tschope D, van de LJ, Clemetson KJ: Thrombospondin binds normally to glycoprotein lllb deficient platelets. Biochem. Biophys. Res. Commun. 179: 985-991, 1,991
• Kehrel B, Flicker E Thrombospondin in Pathophysiology - Thrombospondin in Relation with Disease Processes, in Lahav J (ed): Thrombospondin. Boca Raton, CRC Press, 1 993, pp 1 99-207
• Glycoprotein VI is a major Collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein llb / llla, and von Willebrand factor do not. Blood 91: 491-4991, 1,998
• Patthy L Detecting distant homologies of mosaic proteins. Analysis of the sequences of thrombomodulin, thrombospondin complement components C9, C8 alpha and C8 beta, vitronectin and plasma cell membrane glycoprotein PC-1. J. Mol. Biol. 202: 689-6969, 1 988
• Roberts DD Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J. 1 0: 1 1 83-1 1 91, 1 996

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• 2000
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• 2001
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